Tyr-phosphorylation of PKC-delta and myocyte function

描述（由申请人提供）：本提案的总体目标是确定酪氨酸残基磷酸化在心肌组织中蛋白激酶C δ（PKCd）调节中的作用。有待检验的假设是，PKCd的激动剂特异性激活涉及酪氨酸磷酸化，酪氨酸磷酸化修饰PKC肌丝底物特异性和激酶活性。实验将表征各种刺激对PKCd的酪氨酸磷酸化状态以及对肌细胞机械功能的影响。此外，将确定铰链、假底物和激活环结构域中酪氨酸残基被不可磷酸化或假磷酸化氨基酸取代对PKCd定位和底物特异性的影响。最后，实验将解决PKCd激活是否促进参与调节病理生理过程（肥大或凋亡）的其他信号转导途径的活性。令人信服的初步证据表明PKCd的特定酪氨酸残基的磷酸化改变底物特异性，特别是在肌钙蛋白（Tn）复合物组分。此外，磷酸化酪氨酸（pY）可能会改变激酶的辅因子的要求，使脂质独立的活动。结果将提供新的信息，刺激特异性PKCd激活和活动，以及对心肌肌丝磷酸化和心肌细胞功能的影响。

DESCRIPTION (provided by applicant): The general objective of this proposal is to define the role of tyrosine residue phosphorylation in the regulation of protein kinase C delta (PKCd) in myocardial tissue. The hypothesis to be tested is that agonist-specific activation of PKCd involves tyrosine phosphorylation which modifies PKC myofilament substrate specificity and kinase activity. Experiments will characterize tyrosine phosphorylation status on PKCd with various stimuli and the impact on myocyte mechanical function. In addition, the effect of tyrosine residue replacement with unphosphorylatable or pseudo-phosphorylated amino acids in the hinge, pseudosubstrate, and activation loop domains on PKCd localization and substrate specificity will be determined. Finally, experiments will resolve whether PKCd activation promotes the activity of other signal transduction pathways involved in regulation pathophysiological processes (hypertrophy or apoptosis. Compelling preliminary evidence indicates phosphorylation of specific tyrosine residues of PKCd alters substrate specificity particularly in respect to troponin (Tn) complex components. In addition, phosphorylated tyrosine (pY) may change the cofactor requirements of the kinase enabling lipid independent activity. Results will provide novel information regarding stimuli specific PKCd activation and activity as well as the effect on cardiac myofilament phosphorylation and myocyte functional capabilities.